Project Summary Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membrane (OM) of Gram- negative bacteria such as Escherichia coli, Salmonella typhimurium and many other important pathogens. LPS (or endotoxin) is essential for survival in this large class of bacteria and serves as a first line of defense against hostile environments encountered during host infection. Given the essential role of LPS in the survival of Gram- negative bacteria ? i.e., the bacterial cells die if any step of LPS transport does not occur ? and the unique cell surface it creates, a detailed understanding of the proteins and mechanisms involved in LPS synthesis and transport will be the foundation on which to develop novel antibiotics against these promising new drug targets. Seven proteins make up the LPS transport (Lpt) system: the inner membrane (IM) ABC transporter LptB2FG, the membrane-anchored periplasmic protein LptC, the periplasmic protein LptA, which is speculated to form a bridge between LptC and LptD to protect the hydrophobic acyl chains of LPS during transport through the periplasm, and the OM protein complex LptDE that inserts LPS into the outer leaflet of the OM. In an exciting advance, the structures of all seven proteins in the Lpt system involved in LPS transport have now been solved. Strikingly, the five periplasmic domains of the Lpt system show remarkable structural homology and the crystal structures provide valuable insights into the mechanism of the essential LPS transport process, yet it is still unknown how LPS is transported across the putative periplasmic Lpt bridge of Gram-negative bacteria. Great progress has been made, including identifying the key players in LPS transport, determining the crystal structures for each of the Lpt proteins, developing the bridge model, and identifying and quantitating the LPS binding site on LptA and LptC, and yet many questions remain regarding the mechanism of transport of such a critical molecule in Gram-negative bacterial physiology. The hypothesis that unfolding/folding events occur in the periplasmic Lpt proteins to move LPS along the periplasmic bridge and that removal of amino acid side chains critical to the stabilization of the protein-protein and protein-lipid interactions will disrupt LPS transport in vivo will be tested through a combination of complementary biophysical techniques, computational studies and in vivo assays. The successful completion of the proposed aims will include the identification and quantitation of the interaction interfaces of the periplasmic bridge assembly for LPS transport in Gram-negative bacteria and the mechanism and quantitation of LPS binding to each periplasmic domain in the Lpt system to yield important insights into the essential LPS transport process in bacteria. The long-term goal of this research is to understand the protein-protein and protein-ligand interactions involved in LPS transport to enable the effective design of novel drugs to selectively inhibit LPS transport in Gram-negative pathogens.